Methods for measuring telomere length

ABSTRACT

Methods and compositions for the measurement of telomere length have application in medical diagnostic, prognostic, and therapeutic procedures. The methods for measuring telomere length include primer extension-based methods and probe-based methods. The primer extension methods involve elongation of telomeric, linker, and/or subtelomeric based primers under conditions such that the telomere serves as a template for primer extension and that the resultant primer extension products can be compared to standards of known length to provide a measure of telomere length. The probe based methods allow for telomere length measurements using DNA from lysed or whole cells and involve hybridizing an excess of probe to all telomeric repeat sequences in the telomere, measuring the amount of bound probe, and correlating the amount of bound probe measured with telomere length.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 08/479,916, filed Jun. 7, 1995, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to methods and reagents for themeasurement of telomere length. The invention has applications in thefields of molecular biology, cell culture technology, and medicaltherapeutics and diagnostics technology.

2. Description of Related Disclosures

Telomeres are specialized nucleoprotein structures at the ends ofchromosomes that are important in maintaining chromosome stability andfunction (Blackburn, 350 Nature 569, 1991; all references cited hereinare incorporated by reference herein). Telomeres function throughprevention of aberrant recombination and degradation at the ends of thechromosomes (Henderson et al., 29 Biochemistry 732, 1990; Bourgain etal., 19 Nucl. Acids Res. 1541, 1991), organization of the sub-nucleararchitecture (Gilson et al., 3 Trends Cell Biol. 128, 1993), andinvolvement in the transcriptional suppression of genes at distal loci(Sen et al., 334 Nature 410, 1988). Telomeres are typically composed ofa tandem repetitive array of a short sequence.

In humans, the telomeres are composed of many kilobases of simple tandem5'-TTAGGG repeats (Moyzis et al., 85 Proc. Natl. Acad. Sci. U.S.A. 6622,1988). These repeats are arranged such that the G-rich strand runs 5' to3' towards the end of the chromosome and sometimes extends beyond the 5'end to generate a single-stranded 5'-(TTAGGG)_(n) overhang, where n istypically 9 to 35 (but n can be more than 35 or less than 9). During DNAsynthesis, the termini of the chromosomes are not fully replicated(Watson, 239 Nature New Biology 197, 1972) by the action of DNApolymerase. Incomplete replication occurs at the 3' end of each of thetwo template strands of the chromosome, because the RNA primer needed toinitiate synthesis in effect masks the 3' end of the template. The RNAprimer is degraded after strand synthesis, and, as there are noadditional sequences beyond the 3' end of the template to which primerscan anneal, the portion of the template to which the RNA primerhybridized is not replicated. In the absence of other enzymes, thechromosome is thus shortened with every cell division. This phenomena isreferred to as the "end-replication problem" and is believed to be a keyfactor in the onset of cellular senescence and aging.

Evidence for this end-replication problem was provided by demonstratingthat, in normal human somatic cells (e.g., fibroblasts, endothelial, andepithelial cells), telomeres shorten by 50-200 bp with each celldoubling (Harley et al., 345 Nature 458, 1990; Allsopp et al., 89 Proc.Natl. Acad. Sci. U.S.A. 10114, 1992). As a consequence, all normal humansomatic cells have a limited capacity to proliferate, a phenomenon thathas come to be known as the Hayflick limit, after which the cells enterreplicative senescence. In human fibroblasts, this limit occurs after50-100 population doublings, after which the cells remain in a viablebut quiescent state for many months. See, Goldstein, 249 Science 1129,1990.

Cellular immortalization (the acquisition of unlimited replicativecapacity) is an abnormal escape from cellular senescence. See, Shay etal., 196 Exp. Cell Res. 33, 1991. Cells can escape from cellularsenescence by adding telomeric DNA to the telomeres to overcome theend-replication problem. Most eukaryotic species utilize a novel enzyme,telomerase, to generate telomeric DNA de novo, thus compensating for,rather than avoiding terminal deletions of telomeric repeat sequences.The enzyme human telomerase can add 5'-TTAGGG repeats to the 3' end oftelomeric DNA, thus extending the DNA and preventing telomereshortening.

Telomerase is a complex of protein components and an integral RNAcomponent. The RNA component of the human enzyme contains a short regioncomplementary to the human telomeric repeat sequence (Feng et al., 269Science 1236, 1995). This complementary sequence allows the telomeraseRNA to serve as a template for the catalytic extension of the 3'telomeric termini (Greider et al., 337 Nature 331, 1989).

Cycles of elongation and translocation allow human telomerase to extendprocessively the 3' region of chromosomes with 5'-TTAGGG repeats.

Telomere shortening occurs systematically with each cell division, andtelomerase activation stabilizes telomeres; therefore, knowledge of thetelomere length and the presence or lack of telomerase activity canprovide information about the replicative history and the proliferativepotential of cells. Harley, 256 Mutation Research 271, 1991, suggeststhat telomeres may act as a mitotic clock. The progressive shortening oftelomeres can be viewed as the means by which cells count divisions; asufficiently short telomere(s) can signal replicative senescence innormal cells (Wright and Shay, 8 Trends Genetics 193, 1992).

U.S. Pat. No. 5,489,508, issued Feb. 2, 1996; PCT Pub No. 95/13381,published May 18, 1995; and PCT Pub. No. 95/13382, published May 18,1995, describe, inter alia methods by which the length of telomeres canbe measured.

One approximate measure of telomere length, the length in nucleotides ofthe sum of all telomeric repeat sequences, is the length of a "terminalrestriction fragment" (TRF). The TRF is defined as the length (oraverage length) of fragments generated by complete digestion of thegenomic DNA with a restriction enzyme that does not cleave nucleic acidscomposed entirely of tandem arrays of the specific telomeric repeatsequence of interest. These large fragments can, depending on therestriction enzyme used and the source of the telomeric DNA, compriseboth telomeric repeats and also "subtelomeric" DNA. Subtelomeric DNA iscomposed of DNA sequences adjacent to the tandem telomeric repeatsequences and generally contains telomeric repeat sequences interspersedwith variable telomere-like sequences (Cross et al., 18 Nucl. Acid Res.6649, 1990; deLange et al., 10 Mol. Cell Biol. 518, 1990; Brown et al.,63 Cell 119, 1990). Mean TRF length can provide a measure of telomerelength of telomeres in a cell or a cell population.

TRF length measurement entails digesting genomic DNA with a restrictionenzyme, typically one with a four-base recognition sequence (e.g., AluI,HinfI, MspI, RsaI, and Sau3A), used individually or in combination. Thisdigestion results in the production of short fragments of non-telomericDNA and longer fragments of telomeric DNA. The digested DNA iselectrophoresed, and a Southern blot is performed by hybridizing the DNAto a radiolabeled telomeric probe, such as for human telomeres,5'-(TTAGGG)₃ or 5'-(CCCTAA)₃. The telomeric DNA can then be visualizedby autoradiography and mean lengths of terminal restriction fragmentscalculated from densitometric scans using computer programs known in theart. See, Harley et al., 345 Nature 458, 1990.

Another method for telomere length measurement (see PCT Pub. No.95/13882, supra) involves the synthesis of DNA complementary to thetelomeres of genomic DNA. The synthesized DNA can be labeled orunlabeled, and the length of this DNA can be determined by gelelectrophoresis or other techniques known in the art. Alternatively,telomere length can be measured by the "anchored terminal primer"method, or by a modified Maxam-Gilbert reaction (see PCT Pub. No.95/13382, supra). These two techniques provide for a more directmeasurement of telomere length by exclusion of "the subtelomeric region"in the analysis.

Telomere length serves as a biomarker for cell turnover. Thus,information on the relative age, proliferative capacity, and othercellular characteristics associated with telomere and telomerase statuscan be obtained by measuring telomere length. Measurement of telomerelength can be used to diagnose and stage cancer and other diseases aswell as cell senescence. Other applications for telomere lengthmeasurement include determining the efficacy of treatment with atelomere length modulating compound (Feng et al., 269 Science 1236,1995); discovering agents that modulate telomere length, telomeraseactivity, or the rate of telomere loss; and determining the presence oftelomerase activity.

There remains a need for more rapid, reliable, accurate, and efficientmethods for measuring telomere length so that the full potential of suchapplications can be realized. This invention meets this and other needs.

SUMMARY OF THE INVENTION

The present invention provides improved methods for measuring telomerelength. The methods of the invention can be performed rapidly andprovide increased sensitivity, efficiency, reliability, and accuracy.Moreover, these methods are amenable to automation and high through-putformats and provide, in some embodiments, a means to measure thetelomere length of an individual chromosome, to compare interchromosomalvariance in telomere length, and to measure telomere length of aspecific cell population within a mixture of cells. In addition, themethods allow one to sort cells and/or chromosomes on the basis oftelomere length. The present invention provides numerous advantages overthe conventional method of telomere length measurement.

In one aspect of the invention, a method for measuring telomere lengthis provided that comprises the steps of:

(a) covalently attaching an oligonucleotide linker to a telomere forwhich a measure of length is desired;

(b) contacting a primer comprising a sequence sufficiently complementaryto said linker to hybridize specifically thereto under conditions suchthat said primer extends to form a primer extension productcomplementary to said telomere; and

(c) correlating telomere length with primer extension product size,thereby providing a measure of telomere length.

In one embodiment, the method involves replication or amplification ofthe telomere sequences by, for example, "polymerase chain reaction"(PCR) amplification. A product defined by extension of two primers, a"forward" primer complementary in sequence to the linker covalentlybound to the 3' end of the telomere and a second primer complementary toa subtelomeric region of the chromosome, is exponentially amplified bythis method. This method provides an accurate and sensitive measurementof the telomere length. In a preferred embodiment of this method, adouble-stranded oligonucleotide linker is used, and prior to theligation of the linker, the chromosomal DNA is treated with a nucleaseto generate blunt ends to improve ligation. Those of skill in the artwill recognize that the use of two primers for the extension stepprovides for exponential amplification but that linear amplification,with a single primer, can also be used to determine telomere length inaccordance with the method of the invention.

Another embodiment of the primer extension method of the inventionprovides a rapid means for measuring telomeres using only one primer. Inthis method, a primer complementary to the covalently bound linker isextended using a polymerase and either (i) only those nucleotidescomplementary to the nucleotides in the telomeric repeat; or (ii) thosenucleotides and a nucleotide analog known as a chain terminator, such asa dideoxynucleotide. One or more of the nucleotides can be labeled. Forhuman telomeres, exclusion of dGTP and/or addition of dideoxy GTP(ddGTP) nucleotide results in termination of primer extension at thefirst C nucleotide relative to the 3' end of the G-rich strand of thechromosome. Denaturation and repeated cycles of primer extension anddenaturation result in multiple copies of the telomeric region. One thenmeasures the size of the extension products to estimate telomere length.For example, if one uses a labeled nucleotide, and the label is aradioactive label, one can measure telomere length by correlatingscintillation counts of labeled nucleotide incorporated into primerextension products with telomere length.

In another aspect of the invention, one can optionally dispense with thelinker altogether. In one alternate embodiment of this method, asubtelomeric primer is used as the sole primer, eliminating the need forligating or otherwise covalently attaching a linker to the 3' end of thetelomere. As noted above, repeated steps of primer extension anddenaturation generate multiple single-stranded copies of the telomericregion. In another alternate embodiment, nucleotide analogs known aschain terminators are employed in the primer extension step. This methodcomprises the steps of:

(a) contacting double-stranded chromosomal DNA in a sample with a primerhaving a sequence sufficiently complementary to a 3' end of a telomereto hybridize therewith in the presence of a mixture of nucleotides and adideoxynucleotide under conditions such that said primer extends to forma primer extension product terminating with said dideoxynucleotide; and

(b) correlating telomere length with primer extension product size toprovide a measure of telomere length.

In this embodiment, the use of a specific dideoxynucleotide in theprimer extension step provides a means to replicate only the telomericportion of the chromosome. The dideoxynucleotide selected depends on thetelomeric repeat sequence and the particular strand of the telomere thatwill serve as the template for primer extension. One selects adideoxynucleotide that will not be incorporated until the primer hasbeen extended past the telomeric region. Incorporation of a labelednucleotide or dideoxynucleotide into the extension product, orprobe-based identification of the extension products on a gel, providesa means to determine extension product size, which correlates withtelomere length.

In another aspect of the invention, one can avoid the use of labelednucleotides and gel electrophoresis by employing labeled probes tomeasure telomere length. This method comprises the steps of:

(a) contacting denatured chromosomal DNA with a labeled probe having asequence complementary to a telomere repeat sequence under conditionssuch that said probe hybridizes specifically to telomeric DNA;

(b) measuring amount of bound probe; and

(c) correlating said amount of bound probe measured with telomerelength.

As noted above, this method does not require the use of gels, as in theconventional assay for telomere length determination, and is conduciveto high through-put or automated processes, which is especially usefulfor clinical applications. In a preferred embodiment, the analysis oftelomere length utilizes imaging techniques that allow for not onlyintercellular and intracellular telomere length determination andcomparison, but also the separation of cells or chromosomes based ontelomere length.

The methods of the invention are broadly applicable to the measurementof telomere length in any sample from any origin. The methods areespecially useful and applicable to the measurement of telomere lengthin samples of biological material obtained from humans. Such sampleswill contain cells or cellular materials and will typically be obtainedfrom humans for the purposes of determining remaining proliferativecapacity or lifespan of the cells in the sample, diagnosing medicalconditions, or identifying disease or proliferative states. These andother aspects of the invention are described in more detail below,beginning with a brief description of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a PCR-based telomere length measurement using alinker ligated or attached by tailing to the telomere, a primercomplementary in sequence to the linker, and a subtelomeric primer.

FIG. 2 graphically represents the results of probing different amountsof DNA with a telomere-specific probe using the modified dot-blot method(see Example 1). The graph shows that the signal increased withincreased amount of telomeric DNA.

FIGS. 3A and 3B show a comparison of mean TRF length (in b) analysis bythe conventional method (FIG. 3A) for S2C and BJ cells at variouspopulation doubling levels (PDL), from higher PDL (lanes 1 and 8, FIG.3A) to lower PDL (lanes 7 and 18, FIG. 3A), with the slot-blot method(FIG. 3B), a variation of the dot-blot method. In FIG. 3B, each rowshows the amount of probe bound for cells at a certain PDL or to controlDNA (λHdIII+Sau3A is the negative control and Rep4 is the positivecontrol) of varying amounts, as shown in the key to the Figure, all asdescribed in more detail in Example 2.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides improved methods for measuring telomerelength. Telomeres are nucleoprotein structures at the ends ofchromosomes and have been shown to function in chromosomalstabilization, position, and replication. Telomeres are also believed toserve as the mitotic clock for signaling cellular senescence. Becausechromosomes of normal somatic cells have been shown to lose about 50-200nucleotides of telomeric sequence per cell division, telomere lengthmeasurement provides a means for determining the proliferative lifespanof a cell. Numerous diseases are characterized by accelerated cellproliferation (hyperproliferative states) or decreased proliferativecapacity. Therefore, improved telomere length measurements help meet theneed for improved diagnostic and prognostic methodology.

To facilitate understanding of the invention, the disclosure of theinvention is organized in sections as follows. First, a definitionsection is provided to define terms and phrases used commonly throughoutthe disclosure. This definition section includes a comprehensivedescription of the types of samples, primers, probes and labels that canbe used with the invention. The next section describes methods of theinvention for measuring telomere length. The methods for measuringtelomere length are divided into two major categories, primer extensionbased methods and probe based methods. The probe based methods sectionis further subdivided to describe telomere length measurements using DNAfrom lysed cells and using whole cells. Then, various applications ofthe invention are described; this description is followed by detailedexamples illustrating the invention.

DEFINITIONS

To assist in the understanding of the invention, the following terms asused herein are defined below.

"Abnormal chromosome" means a chromosome which has undergone a deletion,addition, or translocation such that the telomeric region is adjacent tochromosomal DNA not normally adjacent to the telomere.

"Blot" means a DNA-binding filter or substrate, such as nitrocelluloseor a Silent Monitor™ Biodyne B membrane.

"Branched DNA probe" or "bDNA probe" means a probe designed for branchedDNA signal amplification (Urdea, 12 BioTech. 926, 1994; U.S. Pat. No:5,124,246), which involves amplification of the signal produced uponprobe hybridization to a target nucleic acid. The bDNA probe iscomprised of a hybridizing portion complementary to the telomericrepeats (e.g., 5'-(CCCTAA)_(n) -3' or its permutations, where ncomprises 8 or more nucleotides in length, preferably 12 to 15 to 20 ormore nucleotides in length), and so hybridizes with telomeric nucleicacid. The probe further comprises a branched region that providesmultiple secondary probe binding sites. After washing to remove unboundprobe, a labeled secondary probe specific for the branches of the bDNAis hybridized to the bDNA and is detected via the label. The signalincreases in direct proportion to the secondary probe-accessible-siteson the bDNA molecule; thus a rare population of target nucleic acids canbe detected by bDNA hybridization. Sensitivity can be further enhancedby probing the telomeric-repeat-complementary-bDNA with a secondary bDNAprobe specific for the branches of the primary bDNA probe (and atertiary probe specific for the secondary probe, and so on), therebypresenting more numerous hybridization sites for the labeled probe. PNAprobes as well as other modified nucleic acid robes, can also be used asbDNA probes.

"Change in telomere length" means that the average or mean telomerelength of chromosomal DNA in a particular cell population or sample isincreased or decreased relative to other normal somatic cells in anindividual or relative to normal somatic cells in other individuals,i.e., those not suffering from a disease condition.

"Label" means a chemical used to facilitate identification and/orquantitation of a target substance. Illustrative labels includefluorescent (e.g., FITC or rhodamine), phosphorescent, chemiluminescent,enzymatic, and radioactive labels, as well as chromophores. Any of awide variety of labeled reagents can be used for purposes of the presentinvention. For instance, one can use one or more labeled nucleosidetriphosphates, primers, linkers, or probes in the methods of theinvention. The term label can also refer to a "tag" that can bindspecifically to a labeled molecule. For instance, one can use biotin asa tag and then use avidinylated or streptavidinylated horseradishperoxidase (HRP) to bind to the tag, and then use a chromogenicsubstrate (e.g., tetramethylbenzamine) to detect the presence of HRP. Ina similar fashion, the tag can be an epitope or antigen (e.g.,digoxigenin), and an enzymatically, fluorescently, or radioactivelylabeled antibody can be used to bind to the tag. For purposes of thepresent invention, the telomeric repeat itself can be a tag. Telomericrepeat binding proteins are known in the art and bind to eitherdouble-stranded or single-stranded telomeric repeats. If the labelingmethod involves the use of a protein, then native or recombinantproteins can be used; typically, such proteins would be purified for useand detected by virtue of a label attached to the particular protein oran antibody specific for the particular protein.

"Linker" means a single- or double-stranded oligonucleotide composed ofnucleotides that is to be ligated to another oligonucleotide or nucleicacid.

"Linker sequence" means the nucleotide sequence of a linker.

"Long polymerase chain reaction (PCR)" means PCR amplificationconditions suitable for amplification of a relatively large nucleic acid(see Cheng, "Efficient PCR of Long Targets", New Horizons in GeneAmplification Technologies: New Techniques and Applications; SanFrancisco, Calif. (1994)); typically, the amplified nucleic acid has alength greater than about 200 nucleotides, but the use of the word"long" is not intended to limit the length of the nucleic acid that canbe amplified.

"Metaphase spread" refers to a cluster of chromosomes that are derivedfrom cells that have been blocked in metaphase as a result of growth inthe presence of a spindle formation inhibitor, such as colcemid. Thecell is treated with a hypotonic buffer to cause swelling and burst upondropping on a surface. Upon bursting, the chromosomes are released outof the cell and disperse onto the surface in clusters. Typically, thechromosomes are spread out on a microscope slide to facilitatevisualization and microscopic analysis.

"Modified rate of telomere loss" means an increase or decrease intelomere loss over a defined time period (e.g., a year) or biologicaloccurrence (e.g., a population doubling) relative to other normalsomatic cells in that individual, or to normal somatic cells in otherindividuals, i.e., individuals not suffering from a disease condition.

"Oligonucleotide" means a molecule consisting of covalently linkednaturally occurring or synthetically constructed nucleotides and/ornucleotide analogs. As used in this disclosure, oligonucleotides aregenerally primers, probes, and linkers composed of deoxyribonucleotides.However, the oligonucleotides of the invention can also be composed ofribonucleotides, modified analogs of ribo- or deoxyribonucleotides(i.e., synthetic or non-naturally occurring), or mixtures of any of thesame. Usually, nucleotide monomers in an oligonucleotide are linked byphosphodiester bonds. However, as will be apparent to one in the art,alternate linkages can be used, including phosphorothioate,phosphorodithioate, phosphoroselenate, phosphorodiselenoate,phosphoranilidate, phosphoroamidate, peptide, and the like linkages. Forexample, a peptide nucleic acid (PNA) is an oligonucleotide with peptidebonds instead of phosphodiester bonds. Because a PNA has no charge, aPNA has a higher binding affinity than a deoxyribonucleic acid.

"Primer" means an oligonucleotide designed to hybridize to a targetnucleic acid and then be extended by the addition of nucleotides or anoligonucleotide. A primer is typically extended by action of apolymerase or ligase. Typically, an oligonucleotide primer will be 8 ormore nucleotides in length, preferably 12 to 15 to 20 or morenucleotides in length.

"Probe" means an oligonucleotide designed to hybridize specifically witha target nucleic acid. Because human telomeres comprise repeats ofsequence 5'-TTAGGG-3', a telomeric probe, unless otherwise indicated,will be identical or complementary to a sequence contained within asequence of two or more such repeats, i.e., the probe will comprise asequence such as 5'-CCCTAA-3', (for an RNA probe, 5'-CCCUAA-3'), or5'-CTAACC-3', for example. Typically, an oligonucleotide probe will be 8or more nucleotides in length, preferably 12 to 15 to 20 or morenucleotides in length.

"Proliferative capacity" means the inherent ability of a cell or cellsin a tissue to divide for a number of divisions (the "Hayflick" limit)under normal proliferation conditions.

"Riboprobe" means a probe comprised of ribonucleotides. A telomericriboprobe can be produced by transcription of multiple, tandem telomererepeat sequences in a recombinant host cell, typically E.coli, asdescribed in Example 1.

"Sample" means a composition of matter comprising a cell or cellextract. The methods of the present invention can be applied to any typeof sample. Samples of particular interest include cell samples such asnormal or diseased tissue samples, e.g., tumor samples, obtained forpurposes of diagnostic or prognostic analysis. For diagnosis, telomerelength measurement may be performed on a particular cell type, on allcells in a tissue (where various cell types may be present), or onextracts of a cell or cells where extract refers to a whole cell extractor a subfraction thereof, such as a specific chromosome from a cell.Typically, but not in all instances, the sample is treated to render thetelomeric DNA in the cells more accessible. The preparation of the DNAcan be accomplished by any of a variety of methods, depending upon themethod for measuring telomere length to be employed.

"Senescent state" means that state in which a cell has lost the abilityto replicate even in the presence of normally appropriate replicativesignals.

"Spot" means the area taken up by a specific fluorescent signal in aFISH analysis image; spot size corresponds to the amount of probehybridized to a telomere.

"Subtelomeric DNA" or "Subtelomeric region" means chromosomal DNAlocated immediately adjacent (100 to 500 bp but can be up to 1 kb) tothe tandem telomeric repeats of the telomeric DNA and generally containstelomeric repeat sequences interspersed with imperfect telomeric repeator other variable sequences.

"Subtelomeric region of an abnormal chromosome" means the chromosomalDNA immediately adjacent (100 to 500 bp but up to 1 kb) to a telomere ofan abnormal chromosome, e.g., the region adjacent to a telomere from adiseased cell, such as those found in α-thalassaemia patients or formedby recombination-based chromosome truncation (Farr et al., 88 PNAS 7006,1991).

"Telomeric DNA" or "Telomeric region" means the chromosomal DNA locatedon the ends of a chromosome consisting of a tandem repeat array of ashort sequence. In humans, the telomere region is composed of5'-TTAGGG-3' repeats and the corresponding complementary sequence. Thetelomeric regions of different organisms differ with respect totelomeric repeat sequence. The telomeric repeat sequence of telomeresfrom a variety of organisms, including human, Tetrahymena, fungi, andnon-human mammals, are known. For instance, Tetrahymena telomeresconsist of repeats of sequence 5'-TTGGGG-3', and the correspondingcomplementary sequences. Consequently, if one is using the presentmethodologies to determine telomere length of telomeres in a sample ofhuman origin, one will employ a probe or primer distinct from thatemployed if the sample is, for example, of fungal origin. Forconvenience, human telomeric region and human telomeric repeat sequencesare typically referred to herein for illustrative purposes. Thisillustrative use is not intended to limit the invention, and those ofskill in the art will recognize that the present methods can be used tomeasure telomere length of telomeres from any organism.

"Terminal restriction fragment" or "TRF" means the length (or averagelength) of restriction fragments that are generated by completedigestion of the genomic DNA with one or more restriction enzyme(s) thatdo(es) not cleave nucleic acids composed entirely of tandem arrays oftelomeric repeat sequences and that comprise both telomeric repeats andsubtelomeric DNA.

"3' end of the telomere" means the single-tranded region of thetelomere; in humans this region is located on the G-rich strand, and iscomposed of a 5'-(TTAGGG)_(n) repeat sequence, where n is typically 9 to35 (but n can be more than 35 or less than 9).

Primer Extension Based Methods for Measuring Telomere Length

The present invention provides methods for measuring telomere length. Inone embodiment, the method comprises the steps of:

(a) covalently attaching an oligonucleotide linker to a telomere forwhich a measure of length is desired;

(b) contacting a primer comprising a sequence sufficiently complementaryto said linker to hybridize specifically thereto under conditions suchthat said primer extends to form a primer extension productcomplementary to said telomere; and

(c) correlating telomere length with primer extension product size,thereby providing a measure of telomere length.

In one embodiment, this method involves contacting the telomere with alinker under conditions in which the linker is ligated by the action ofa DNA or RNA ligase that can "blunt-end" ligate together twosingle-stranded RNA or DNA molecules. This method can also be employedusing a double-stranded linker. Alternatively, the linker can beattached nucleotide-by-nucleotide by a terminal transferase and specificdNTPs (dCTP, dTTP, dGTP or DATP, depending on the sequence to be added).Terminal transferase can add multiple monomers of a specified dNTP tothe 3' end of the telomere; the added nucleotides can then serve as aprimer annealing site.

An oligonucleotide complementary in sequence to the sequence of thelinker is used as a primer in the replication step. This oligonucleotideis referred to as the "forward primer". In addition to the forwardprimer, a second primer can preferably be used. The telomere sequencesare replicated or amplified, for example, by PCR amplification with afirst primer specific for the linker sequence and a second primerspecific for a subtelomeric region of the chromosome. The primers can beextended by any means that requires the presence of the telomeric regionfor primer extension to occur; preferred means are mediated by atemplate-dependent DNA or RNA polymerase, a template dependent DNA orRNA ligase, or a combination of the two. Telomeric nucleic acids in asample are covalently bound to a linker, and a primer complementary tothe linker and/or a subtelomeric primer provide(s) a substrate foreither DNA or RNA polymerase or DNA ligase to produce primer extensionproduct(s) complementary to the telomeric region.

As noted above, if the primer extension reagent is a DNA polymerase, anda second primer is present, one has the requisite components for PCR, aprocess more fully described in U.S. Pat. Nos. 4,683,195; 4,683,202; and4,965,188, provided the appropriate buffer and nucleoside triphosphatesare present in the reaction mixture. Taq polymerase (available formPerkin-Elmer) is a preferred polymerase in PCR amplification, althoughother polymerases, especially thermostable polymerases, can be employed.Taq polymerase can misincorporate nucleotides in the primer extension.If misincorporation presents a problem, and typically, themisincorporation is at such a low frequency that no problem isencountered unless the amplification products are very long, thenanother polymerase with a lower frequency of misincorporation, i.e.,Pfu, Pwo, or Vent can be employed, or a mixture of such polymerases,i.e., Taq polymerase/Vent DNA polymerase in a ratio range of 100:1 to10:1, can be employed. The appropriate selection of a polymerase or apolymerase mixture can provide optimal extension of the primer extensionproduct.

Once a primer extension product has formed, one can disassociate ordenature (typically by heat denaturation, but other methods, i.e.,enzyme or chemical mediated processes, such as helicase mediateddenaturation, can be used) the primer extension product from thetelomeric region. If additional primer and primer extension reagent ispresent in the sample, then a new primer/telomere complex can form,leading to the production of additional primer extension products. Onecan repeat the process of primer extension and denaturation several tomany times, as desired. Typically, primer extension and denaturation ofextended primer/telomere complexes will be performed at least 5, 10, 15,20, to 30 or more times. Moreover, if a second primer complementary tothe subtelomeric region of the extended primer is present in thereaction mixture, one can increase the replication products (bothextended primer and the complementary extended sequence) dramatically,because the two primers mediate PCR amplification of the telomere.

The PCR cycles are composed of cycle times and temperatures that canvary widely, depending upon the sample, detection format, andapplication. Typically, long PCR reaction conditions are employed. Thesimplest PCR cycle comprises a duplex nucleic acid denaturation stepfollowed by a primer annealing and extension step. The denaturation steptypically involves heating at any of a relatively wide range oftemperatures for an amount of time sufficient to denature but not damagethe DNA. In similar fashion, the time and temperature of the primerannealing step depend on the reaction buffer and primer sequence,concentration, and composition, as well as the specificity required bythe practitioner. The time and temperature of the primer extension stepdepend upon the type of DNA polymerase or ligase employed. Those ofskill in the art will recognize and understand that the presentinvention is not limited by the times, temperatures, and reactioncondition variations in buffer and other reaction components that can beemployed.

The second primer used in this embodiment is a primer comprisingsequences complementary to the subtelomeric region and is designated the"subtelomeric primer." FIG. 1 illustrates this method, showing Pi as thesubtelomeric primer, P2 as the forward primer, and the zigzag lineindicates the junction between the subtelomeric region and the telomere(the figure is not drawn to scale) . In this Figure, the telomere is ahuman telomere and so comprises the human telomeric repeat 5'-TTAGGG-3'and complementary repeat sequences. The region depicted by heavy rightangled slashed lines in FIG. 1 represents the subtelomeric regioncomplementary to the subtelomeric primer. Preferably, the subtelomericprimer does not comprise a telomeric repeat sequence and so is anon-telomeric repeat sequence of the subtelomeric region. Preferably,the subtelomeric primer anneals to the subtelomeric region within 50base pairs of the junction with the telomeric region. Extension of theseprimers in multiple cycles of primer annealing and extension amplifiesthe telomeric region by producing multiple replicates of the same.

If the primer extension product is generated by PCR, then, as notedabove, one also employs a primer specific for the subtelomeric region.This primer is a site specific and, preferably, a chromosome specificprimer. The subtelomeric primer is complementary to a sequence in thesubtelomeric region that is present in at least one chromosome, butoptionally present in two or more, up to and including all chromosomes,of the cell or cell population of interest. Subtelomeric regions of manydifferent chromosomes are known in the art. Additional subtelomericregions of normal and abnormal chromosomes can be determined usingcloning and sequencing procedures known in the art.

Primers specific for a subtelomeric region of a normal chromosomeinclude novel primers based on the heretofore unpublished TelBam 8 probesequence, specific for chromosome 7q, such as TEL8-1,5'-TGCAATTATTTTACTATCTGTTATCGG-3' (SEQ. ID NO.:1); and TEL8-2,5'-TGACCTGTTTTAAAGAGTATGCTCAG-3' (SEQ. ID NO.:2). Nucleic acidscomprising all or portions of the TelBam 8 probe sequence can be clonedas described in Brown et al., 63 Cell 119, 1990.

Other illustrative subtelomeric primers include XpJCTN,5'-CCCTCTGAAAGTGGACCWATCAG-3' (SEQ. ID NO.:3), where XpJCTN is a mixtureof two primers, in one of which W is A and in the other of which W is T;40BPXpJCTN, 5'-CTTTTATTCTCTAATCTGCTCCC-3' (SEQ. ID NO.:4); 400BPXpJCTN,5'-TAGGGGTTGTCTCAGGGTCCTA-3' (SEQ. ID NO.:5); REV40BPXpJCTN,5'-GGGAGCAGATTAGAGAATAAAAG-3'(SEQ. ID NO.:6); and revXpJCTN,5'-CTGATWGGTCCACTTTCAGAGGG-3'(SEQ. ID NO.:7), which are based onsequences in the pseudo-autosomal region of the X and Y chromosomes(Baird et al. 14 The EMBO Journal 5433, 1995)

By attaching a linker to the 3' end(s) of the chromosome(s), and using aforward primer specific for the linker and a primer complementary to thesubtelomeric region of a chromosome (e.g., a primer incorporating asubtelomeric sequence, such as the sequence of the TelBam 8 probe or thepseudo-autosomal region of chromosomes X and Y), PCR amplification cangenerate multiple copies or replicates of the telomeric region, whichcan in turn be used to measure telomere length. The PCR primer extensionproducts are, for example, separated by size on a gel. If theamplification products have been labeled, i.e., by incorporation of alabeled nucleotide or hybridization to a labeled probe, then one can usesize standards to determine telomere length. Direct incorporation oflabeled nucleotides into the amplification products allows forelimination of the steps comprising denaturing the DNA in the gel,neutralizing the gel, drying the gel, hybridizing a probe to the gel,removing unbound probe from the gel, and exposing the gel, typicallyundertaken in conventional probe-based methods. Relative to conventionalmethods, which can require about a week to complete, the PCR-basedmethod for measuring telomere length is quick, accurate, sensitive, andrequires significantly less sample to perform.

In a preferred embodiment of this method, the linker is adouble-stranded oligonucleotide, and the telomeric DNA is treated priorto linker attachment to remove or fill in single-stranded regions. Inthis embodiment, as exemplified in Example 4, the DNA is treated with anuclease (i.e., Bal31, Mung bean, or other nuclease) and/or DNApolymerase, such as T4 or Pfu (these polymerases possess 3' to 5'exonuclease activity in combination with their 5' to 3' polymeraseactivity), to generate blunt-ended, double-stranded telomere ends priorto ligation of the double-stranded linker.

While those of skill in the art will recognize that any double-strandedlinker can be used, so long as the linker sequence differs from thetelomeric repeat sequence, a particularly preferred double-strandedlinker is composed of complementary single-stranded oligonucleotidesSLIC-II and aSLIC (see Edwards et al., 19 Nucl. Acids Res. 5227, 1991),shown below.

5'-GGAATTCTGGTCGACGGATCCTGA-3' SLIC-II (SEQ. ID NO.:8)

3'-CCTTAAGACCAGCTGCCTAGGACT-5' aSLIC (SEQ. ID NO.:9)

The 5' end of the SLIC-II oligonucleotide can be constructed so as toterminate with a 5'-phosphate, whereas the 3' end of thisoligonucleotide can be constructed with a terminal2',3'-dideoxyadenosine group to prevent the linker from ligating toanother linker. Likewise, the complementary aSLIC oligonucleotide can beconstructed so that the 3' end terminates with a 2'3'-dideoxycytidine toprevent ligation to another linker. A double-stranded oligonucleotideformed by hybridization of SLIC-II to aSLIC so constructed cannotself-ligate.

The double-stranded oligonucleotide linker formed by SLIC-II hybridizingto aSLIC also contains restriction sites to facilitate cloning or otherapplications. This double-stranded oligonucleotide linker can be ligatedto the 3' end of the blunted telomere using a ligase (i.e., T4 DNAligase or other ligase). A primer complementary to SLIC-II, such asaSLIC, 5'-CCGTCGACCAGAATTCC-3' (SEQ. ID NO.:10) or5'-CAGGATCCGTCGACCAG-3'(SEQ. ID NO.:11), can be used to generate thedesired primer extension product. As noted above, the use of a second,subtelomeric primer for extension provides a means for PCR amplificationof the telomeric region. Separation of the PCR amplified primerextension products by size on a gel and visualization of the products,for comparison to a standard(s) of known length(s), provides the lengthof the telomere DNA in the sample.

While the PCR based embodiments of the present invention are quiteuseful, the present method can be practiced using any method of primerextension to provide target amplification or with a method that providesfor signal amplification or both, as described below. Moreover, targetamplification can be achieved by means other than PCR. These methodsinclude the ligase chain reaction (Barany, 88 Proc. Natl. Acad. Sci.U.S.A. 189, 1991), nucleic acid sequence-based amplification (Compton,350 Nature 91, 1991), self-sustained sequence replication (Guatelli etal., 87 Proc. Natl. Acad. Sci. U.S.A. 1874, 1990), and stranddisplacement amplification (Walker et al., 89 Proc. Natl. Acad. Sci.U.S.A. 392, 1992). While PCR and other amplification methods provide forexponential accumulation of primer extension products, even linearaccumulation of primer extension products can provide useful results.Thus, one can use a single primer and merely make many copies of thetelomeric region from this one primer, as described more fully below.

This invention provides a method to measure telomere length using linearamplification. This method exploits the fact that the human telomericrepeat sequence lacks guanidine residues in the C-rich strand; however,this method is generically applicable to telomeres of any origin thatcomprise repeat sequences that lack one or more nucleotides. In thisembodiment, a primer complementary to the covalently bound linker isadded to the genomic telomere DNA in the presence of only three (forhuman telomeres, DATP, dTTP, and dCTP) of the four nucleosidetriphosphates. These three dNTPs form the complement to the G-rich standof a human telomere. Usually, the primer or at least one of thetriphosphates is labeled with a detectable label, e.g. a radioisotope ora fluorescent molecule, which label is retained upon incorporation intothe primer extension product.

The primer is extended by means of a primer extension reagent, e.g., aDNA polymerase such as the Klenow fragment of DNA polymerase I, T7 DNApolymerase, or Taq DNA polymerase or the Stoffel fragment thereof.Exclusion of dGTP results in termination of the primer extension at thefirst C nucleotide of the chromosome. Thus, sequences complementary tothe primer located outside the telomeric and/or subtelomeric regionwould not serve as templates for primer extension products due to thelack of dGTP. Denaturation of the primer extension product from thetelomeric DNA followed by repeated cycles of primer extension results inthe generation of multiple copies or replicates of one strand, for humanchromosomes, the G-rich strands, of the telomeric region. For manypurposes, a simple measure of the label incorporated suffices toquantitate telomere length, although one can also measure the lengthdirectly by gel electrophoresis and comparison to standards of knownlength.

In a preferred embodiment, the annealed primer is extended in thepresence of a mixture of dideoxy GTP (ddGTP), dCTP, DATP, and dTTP by apolymerase (for human telomeres). The method differs from the previousembodiment in that, instead of leaving one or more nucleotides out ofthe reaction mixture, one uses a chain-terminating nucleotide(s) inplace of the otherwise missing nucleotide(s). Polymerization proceedsuntil the polymerase encounters the first cytosine residue (for humans)in the subtelomeric region. The enzyme will then incorporate the ddGTPnucleotide and further extension will be terminated due to the presenceof the ddGTP. The primer extension reaction can repeated as many timesas desired. The length of the extended DNA can be determined by gelelectrophoresis and comparison to standards, as described above. Inaddition, the amount of DNA synthesized can be determined by measuringthe label incorporated into the primer extension products or the amountof probe hybridized to the primer extension products, which will bedirectly proportional to telomere length. While the manner ofdetermining primer extension size may vary depending upon the method ofanalysis selected, any of the methods described can be used.

In another embodiment of this method, linear extension productsgenerated using a subtelomeric primer serve to provide a measure oftelomere length. This embodiment eliminates the need for ligating orotherwise covalently attaching a linker to the 3' end of the telomere.As above, this embodiment is ideally suited for linear amplificationresulting in multiple copies of the telomeric region.

If the subtelomeric primer selected is not specific or unique to thesubtelomeric region, then the primer extension products generated fromthe subtelomeric region can be distinguished from those generated froman internal chromosomal region by hybridizing with a probe that isspecific to a telomeric region or a region immediately adjacent to thetelomeric repeat sequences. Alternatively, if the subtelomeric primerhybridizes to multiple sites within the subtelomeric region, then onecan hybridize a primer specific to a region immediately adjacent to thetelomeric repeat sequences, and this second subtelomeric primer can beannealed to the first primer extension products and extended with DNApolymerase and the size of the second primer extension productsdetermined as described above to provide a measure of the subtelomericlength in the first primer extension products.

Another embodiment of the invention involving the use ofchain-terminating synthetic nucleotide(s), such as a dideoxynucleotide,eliminates the need to attach a linker covalently to the 3' end of thetelomere. This method comprises the steps of:

(a) contacting double-stranded chromosomal DNA in a sample with a primerhaving a sequence sufficiently complementary to the 3' end of a telomereto hybridize therewith in the presence of a mixture of nucleotides and adideoxynucleotide under conditions such that said primer extends to forma primer extension product terminating with said dideoxynucleotide; and

(b) correlating telomere length with primer extension product size toprovide a measure of telomere length.

As noted above, this method can be used as a variation of thelinker-based method, where the primer is complementary to a linker addedto the 3' end of the telomere, but in a preferred embodiment, no linkeris required. In this embodiment, an oligonucleotide sequencecomplementary to the 3' single-stranded region of the telomere, i.e.,5'-(CCCTAA)₄ -3' (SEQ. ID NO.:12), is annealed to the telomeric DNAtermini. The annealed primer is extended in the presence of a mixture ofdideoxy GTP (ddGTP), dCTP, DATP, and dTTP by a polymerase (for humantelomeres). In a preferred embodiment, one of the nucleotides is labeled(typically, in any method involving incorporation of a labelednucleotide, especially a radioactively labeled nucleotide, only a smallfraction of the total nucleotide is labeled, and so the labelednucleotide is referred to as a "tracer"). The polymerization willproceed until the polymerase encounters the first cytosine residue inthe subtelomeric region, as described above. The DNA is then denaturedand separated by size on a gel. Incorporation of a labeled nucleotideprovides for easy identification of the length of the extension producton a gel and direct correlation of telomere length from signalintensity. One can, however, also use a labeled probe to detect primerextension products.

In a more preferred embodiment of this method, the genomic DNA isfragmented with a restriction enzyme that cuts DNA, but not telomericDNA, frequently, i.e., for human chromosomes, HinfI or any otherrestriction enzymes with relatively short recognition sequences, andsubsequently treated with a polymerase (i.e., DNA polymerase I or theKlenow fragment thereof, Taq polymerase or the Stoffel fragment thereof)in the presence of ddGTP, dCTP, dATP and dTTP, prior to addition of theprimer. This treatment fills in nicks and gaps in the genomic DNA andblocks any potential priming sites in the genomic DNA with a ddGTP chainterminator. Pretreatment of the DNA to fill in gaps and nicks can resultin increased sensitivity and decreased background signal. Anoligonucleotide primer with a sequence complementary to the 3' singlestrand region of the telomere is then added and annealed to thetelomeric DNA termini and extended as discussed above. As before,telomere length is determined by correlation to the size of the primerextension products.

The foregoing methods involve the use of a primer and the detection ofprimer extension products to measure telomere length. The following twosections describe methods using a probe to measure telomere length.

Probe Based Methods for Measuring Telomere Length

In another aspect of the invention, labeled probes are employed tomeasure telomere length. This method comprises the steps of:

(a) contacting denatured chromosomal DNA with a labeled probe having asequence complementary to a telomere repeat sequence under conditionssuch that said probe hybridizes specifically to telomeric DNA;

(b) measuring amount of bound probe; and

(c) correlating said amount of bound probe measured with telomerelength.

In these probe-based methods, the probe is added in excess, so that allor substantially all of the telomeric repeats in the telomere arehybridized to the probe. Typically, the correlation step involves theuse of standards of known length or the use of conversion factors toconvert the amount of bound probe to a measure of telomere length.

This aspect of the invention provides a method for measuring telomerelength in which an oligonucleotide probe is hybridized to telomererepeat sequences. The amount of probe hybridized is determined and thencorrelated to provide a measure of telomere length. This method can bepracticed without the gel based size separation step used in othermethods. Thus, this aspect of the invention provides a rapid, highthrough-put method for measuring telomere length.

In a preferred embodiment, this method involves preparing DNA extractsof cells, incubating the extract with an oligonucleotide probecomplementary to telomere repeat sequences, and determining amount ofprobe bound as a measure of telomere length. For convenience, the cellscan be grown in a 24, 48, or 96-well microtiter plate.

To practice the method, the cells from each well are collected and theDNA isolated by standard DNA extraction procedures. The DNA extractsolution can be passed through a DNA-binding filter, such asnitrocellulose or Biodyne B membrane, to remove other potentiallyinterfering substances. The filter is then contacted with a labeledoligonucleotide probe having a sequence complementary to telomere repeatsequences. After unbound probe is removed, the amount of probe bound tothe filter is then quantified, and the amount of probe bound provides ameasure of telomere length. This method, called the dot-blot method, isexemplified in Example 1 below. As with other embodiments, standards ofknown telomere length can be employed to help correlate the signal frombound probe with telomere length. In a preferred embodiment, this methodis carried out in a 24, 48, or 96-well plate and is automated.Preferably, Pall SILENT MONITOR™ 96-well test plates, having 0.4 μMBiodyne B membrane located at the bottom of each well, are used.

In another embodiment, the invention provides a method of measuringtelomere length in which the genomic (chromosomal) DNA is bound to asolid phase using a modified dot-blot method called the slot-blot. Thisaspect of the invention is illustrated in Example 2. The slot-blot isdescribed for a distinct application in Kafatos et al., 7 Nucl. AcidRes. 1541, 1979, and has been used to determine the relativeconcentrations of nucleic acids in a mixture. A distinct DNA to membranecross-linking step and unique filtration apparatus distinguish thismethod from the dot-blot method described above. In this embodiment ofthe invention, samples of nucleic acid (i.e., genomic DNA) are spottedon and cross-linked to a nitrocellulose filter (e.g., Schleicher &Schuell nitrocellulose filter) using UV irradiation, and the nucleicacid on the filter is hybridized with a labeled oligonucleotide probe.Typically, the genomic DNA is sheared or cleaved into smaller fragmentsprior to binding to the solid phase. The genomic DNA is cleaved orsheared enzymatically or mechanically, i.e., with restrictionendonucleases, sonication, or other methods known in the art. In apreferred method, the cleaved DNA is probed with a riboprobe. The amountof probe hybridized to the nucleic acid in each of the slots isquantitated, and the quantitated amount is correlated with telomerelength, e.g., by comparing to a standard.

In another embodiment, one measures the loss of or decrease in boundprobe observed after treatment of the genomic DNA with a known amount ofexonuclease that degrades DNA specifically from the ends of thechromosome to measure telomere length. A preferred exonuclease is Bal31,an exonuclease that digests single- or double-stranded DNA specificallyfrom the end of a DNA, such as the end of a telomere. The rate of Bal31digestion is about 50 bp/min. Thus, when chromosomal DNA is digestedwith Bal31, DNA internal to the telomeres is digested last whiletelomeric DNA is digested first. The method can be conveniently carriedout by spotting Bal31 enzyme (i.e., serial dilutions) on a DNA bindingmembrane, i.e., nitrocellulose, located on the bottom of each well of aplate, binding genomic DNA to the membrane, incubating under conditionswhere the nuclease enzyme is active for a specific period of time,denaturing the enzyme and DNA, and hybridizing the remaining DNA to atelomeric probe, i.e., for human DNA, a probe comprising 5'-TTAGGG-3'repeats, under hybridizing conditions. The amount of probehybridization, which should decrease with increasing Bal31 concentrationor reaction time, can again be used to determine the telomere length.Preferably, this method is carried out in a multi-well, i.e., 96-well,format and, more preferably, is automated. If desired, telomeres ofknown length or cells comprising telomeres of known length can be usedas standards.

Telomere Length Measurement in Whole-Cells

The methods of the invention can be applied to whole cells, as well ascell extracts. In one embodiment, whole cells are attached to a solidsupport or surface; the cells are permeabilized; the cellular DNA isdenatured; and a labeled telomere probe (or a mixture of labeled probes)is added and hybridized to the telomeric repeats in the denatured DNA.In preferred embodiments, a fluorescein tag and "anti-fade" agents, asdescribed below, are used, and the results of this "fluorescence in situhybridization" (FISH) are analyzed using confocal microscopy. See Trasket al., 91 Proc. Natl. Acad. Sci. U.S.A. 9857, 1979, incorporated hereinby reference.

In a preferred embodiment, this method is used to measure telomerelengths of chromosomes in a metaphase spread. Chromosomes are stained orlabeled with a chromosomal dye (e.g., DAPI/DA); the slides arepreferably prepared using antifade mounting medium (e.g. 9:1glycerol:PBS containing 0.1% p-phenylenediamine buffered to pH 8.0 with0.5M carbonate/bicarbonate buffer). The fluorescent signal is preferablyamplified. The results can be analyzed with an image generator inconjunction with an inverted fluorescence microscope.

FISH analysis of cells or metaphase spreads of cells can be used for avariety of purposes: to determine average relative telomere lengths incells in a tissue sample; to determine the longest telomere length incells in a sample; to detect the presence of certain types of cells,i.e. certain stem cells can be identified by their long telomeres; todetermine the size distribution of telomere lengths in a sample; todetermine changes in telomere length in a cell population over time orafter treatment with an agent or exposure to certain conditions; and todetect different cell types within a tissue, i.e., cancer cells can beidentified by their having telomeres of a different length than thatobserved in normal cells, such as cells adjacent to tumor cells.

Quantitative FISH analysis with confocal microscopy using signalintegration also allows one to obtain an objective measure of thedistribution of telomere lengths on different chromosomes and toidentify chromosomes which have lost a critical amount of telomeric DNA,indicative of the presence of aberrant cells. The method requires onlyrelatively small samples and allows for direct measurement of telomerelength on a chromosome-by-chromosome and cell-by-cell basis. Theintensity of signal from bound probe per chromosome or cell isproportional to the number of telomeric repeats, and thus to thetelomere length. The method also provides a means to investigatetelomere heterogeneity in cell or tissue samples; such information canbe especially useful when combined with information regarding thepresence and amount of telomerase activity in the sample (Harley et al.PCT Pub. No. 95/13381, published May 18, 1995; Harley et al. U.S.application Ser. No. 08/631,554, filed Apr. 12, 1996; and Harley et al.U.S. application Ser. No. 08/632,662, filed Apr. 15, 1996).

Many variations of in situ hybridization can be applied in the methodsof the invention. For example, a variation of the in situ hybridizationdetection method involves primed in situ labeling ("PRINS"; Koch, J., in"Nonradioactive in situ Hybridization Application Manual" (1992),Boehringer Mannheim, 31-33). This method involves the use of a primerbut is discussed in this section for ease of understanding. Detection oftelomere repeats by PRINS involves using an oligonucleotide primerspecific for telomere repeats and chain elongation incorporating labelednucleotides. In a typical protocol, a PRINS reaction mixture (10 μl) of5% (v/v) glycerol; 10 mM Tris-HCl, pH 8.3; 100 mM KCl; 0.05% (w/v) Tween20; 0.75 mM EGTA; 2.5 mM MgCl₂ ; 0.4 μM return primer; 200 μM DATP,dGTP, dCTP; 110 μM dTTP; 90 mM labeled dUTP is placed on a fixed,permeabilized sample, sealed with a coverslip, anchored with nailpolish, overlayed with mineral oil, and incubated at 70° C. for 30minutes to 3 hours. After completion of the PRINS, the sample is washed3 times in wash buffer (0.6M NaCl and 0.06M sodium citrate (4×SSC);0.05% Tween 20) heated to 70° C. for 2 minutes, and the signal observedas described above. To reduce the background signals that can arise fromdirect incorporation of fluorescent labels during primer extension,indirect detection using unlabeled dNTPs and unlabeled primers can beused and the product detected using a labeled probe.

In an additional preferred embodiment, the method allows one todetermine cellular DNA content simultaneously with measuring telomerelength. Cellular DNA content can indicate whether a cell isproliferating or senescent. If the cell is proliferating, thechromosomal DNA content can increase up to two-fold. Consequently, thesignal intensity of the bound telomeric probe for a rapidlyproliferating cell with short telomeres can be equal to or stronger thanthat of a non-dividing cell with longer telomeres. Thus, this method maybe useful to normalize the measured signal intensity of the telomericprobe with respect to DNA content. The method comprises the steps ofattaching cells or metaphase spreads to a support; denaturing thecellular DNA; contacting the chromosomal DNA with a labeled telomericprobe and a DNA specific dye; hybridizing the denatured DNA with theprobe; and measuring the amount of probe hybridized and cellular DNAcontent simultaneously using flow cytometry. This method allows one todetermine cell cycle position as well as telomere length. The intensityof signal from bound probe per chromosome or cell is proportional to thenumber of telomeric repeats, and thus to the telomere length. Oneadvantage of this method is that cells can then be sorted, e.g., using aflow cytometer (Coulter EPICS ELITE fluorescence activated cell sorter(FACS)), based on telomere length. The instrument can be programmed todeflect the cells into specific tubes based upon telomere length.

As noted above, the invention provides for the measurement of the lengthof a telomere of an individual chromosome. Flow cytometry facilitatesthis analysis. A combination of fluorescent dyes (e.g., chromomycin A3,a major or minor groove binding dye with relative GC specificity, andbisbenzimide 33242, a groove binding dye with relative AT-specificity)in conjunction with a probe hybridized to the telomeric region, can beused to direct the flow cytometer to analyze and sort isolated metaphasechromosomes by telomere length. The chromosomes can be labeled to obtaina direct measure of the telomere length of an individual chromosome andsubsequently sorted using a three-laser flow cytometer. The measurementis made by quantifying the fluorescent intensity for each individualchromosome using flow cytometry analysis. Alternatively, simultaneousthree-color staining methods, in which the chromosomes are prepared,sorted, and subsequently hybridized in solution, can be applied totelomere length analysis of individual chromosomes.

The methods of the invention can be performed rapidly and provideincreased sensitivity, efficiency, reliability and accuracy. Moreover,these methods for telomere length measurement can be employed in a highthrough-put and/or automated process format. These telomere lengthmeasurement methods can be used for diagnostic, prognostic, and researchapplications.

Applications

Telomere length measurement has useful application in medicaldiagnostics, prognostics, and therapeutics. Such applications include,but are not limited to: (I) determination of the proliferative lifespanof cells; (ii) identification and analysis of the effectiveness ofagents capable of extending, maintaining, or reducing telomere length;(iii) diagnosis of disease or medical conditions characterized by adifferent telomere length in a patient relative to an individual nothaving the disease or particular medical condition; (iv) prognosis ofdisease or medical conditions as correlated to telomere length; and (v)identification of cells, cell types, or cell populations. In general,measurement of telomere length provides a powerful means to assess andmonitor cellular lifespan for a variety of useful purposes.

The length of the telomeres of the chromosomes in a cell is indicativeof the proliferative capacity of that cell, and so provides an indicatorof the health of an individual or organism comprising such cells.Certain populations of cells may lose telomeres at a greater rate thanthe other cells within an individual. Rapid and/or extendedproliferation of those cells may make that cell population age-limitedor senescent, with negative impact on an individual relying on that cellpopulation for health. The diagnostic procedures described herein can beused to indicate the potential life span of any cell type, as well as tofollow telomere loss over time, so that revised estimates of life spancan be made over time.

Telomere length measurement can be used to monitor the effectiveness ofvarious therapeutics in expanding and/or reducing the proliferativelifespan of cells. In one example, cells treated with an oligonucleotidecomprising telomeric sequences had a reduced rate of telomere loss andan increased proliferative capacity of about 10 population doublings.Conversely, the treatment of cells with AZT or other small organicmolecule inhibitors of telomerase can increase telomere loss and reducethe proliferative capacity of the treated cells. Telomere lengthmeasurements facilitate the analysis of the efficacy of such agents oncells. See U.S. Pat. No. 5,489,508, issued Feb. 2, 1996.

Telomere length measurement can also be used to monitor theeffectiveness of cancer chemotherapeutics during treatment. Telomerelength measurement provides a means to determine the effectiveness of atelomerase inhibitor or other agent (i.e., a retinoid) that repressestelomerase expression, because telomere length will decrease over timein dividing cancer cells in which there is inhibition of telomerase ortelomerase expression. Measuring the telomere length of chromosomes intumor cells can provide information regarding the proliferative capacityof such cells, both before and after administration of telomeraseinhibitors or other treatments that affect telomere length. In a relatedapplication, one can measure the telomere length of telomeres inhematopoietic stem cells (HSCs) such as CD34+ cells, prior to use inbone marrow transplantation. The longer the telomeres, the more likelythe cells will successfully engraft.

The methods of the invention are also generally useful in discoveringagents that modulate telomere length. Cells can be treated with testagents (e.g., synthetic compounds, fermentation extracts, nucleic acidpreparations, and other agents) during culture to determine the effectof such test agents on telomere length and telomere maintenance ofspecific chromosomes.

In diagnostic applications of the invention, telomere length measurementcan detect a change in telomere length and/or the rate of telomere loss.A tissue can have a spectrum of cells of different proliferativecapacity. Average telomere length for a tissue will be informative ofthe state of the tissue generally. Multiple measurements of telomerelength over time can be used to determine the rate at which the telomerelength changes over time.

In addition, telomere length measurement methods of the invention can beused to diagnose the presence of abnormal chromosomes. If one uses aprimer specific for a known subtelomeric region of an abnormalchromosome, such as chromosome 6 from cells of α-thalassaemia patients,the primer based methods of the invention can be used to diagnose thedisease states associated with such cells. The presence of primerextension products is indicative of the presence of the abnormalchromosome indicative of the disease state.

In prognostic applications of the invention, telomere length measurementcan detect whether a cellular disease, such as cirrhosis of the liver ormuscular dystrophy, has affected the proliferative capacity of thediseased tissue so as to impact the recuperative capacity of thepatient. In other situations, such as those involving injury to atissue, as in surgery, wounds, burns, and the like, the ability ofcells, e.g., fibroblasts, to regenerate will be of interest, andtelomere length, a function of proliferative capacity, provides suchinformation. Similarly, in the case of bone loss, osteoarthritis, orother disease requiring reformation of bone, the renewal orproliferative capacity of osteoblasts and chondrocytes will be ofinterest, and again, telomere length provides an indicator ofproliferative capacity. In addition to cellular diseases, diseasesassociated with aging can be diagnosed using the present methods. Inthese applications, telomere length provides an indicator ofproliferative capacity, because the longer the telomere of a cell, thegreater the potential replicative capacity that cell possesses.

A variety of diseases and disease states are amenable to diagnostic andprognostic evaluation by telomere length measurement. For example, thereis a reduction in telomere length and replicative capacity infibroblasts from patients with the accelerated aging syndromeHutchison-Gilford progeria relative to age-matched normal individuals(Allsopp et al., 89 Proc. Natl. Acad. Sci. U.S.A. 10114, 1992).Accelerated telomeric loss is also associated with immunosenescence,such as that occurring prematurely in lymphocytes of individuals withDown's Syndrome (DS). DS patients show many features of premature aging,and lymphocytes from DS patients lose telomeres at three times the rateof age-matched controls (Vaziri et al., 52 Am. J. Hum. Genet. 661,1993). Accelerated cellular turnover and concomitant telomere loss percell division correlate with the premature aging phenotype, and telomereloss or short telomeres in immune cells is a biomarker ofimmunosenescence. Any disease associated with a higher rate of cellturnover or division is amenable to diagnosis and prognosis with thepresent invention.

For example, atherosclerosis in part results from a higher rate of cellturnover in the intimal and medial tissue in areas of atheroscleroticplaque relative to the surrounding normal tissue. Cells derived fromthese regions of atherosclerotic plaque undergo more cellular divisionsthan cells from plaque-free areas, in effect rendering the cells inplaque areas older and nearer to the end of their maximum replicativelifespan. Telomere length serves as a biomarker of cell turnover intissues involved in atherosclerosis. In general, telomere loss inintimal and medial tissue underlying an atherosclerotic plaque isgreater than that in plaque-free regions.

Formation of atherosclerotic plaques occurs more often in the iliacartery than in the iliac vein, and as expected, the decrease in mean TRFlength in one test was shown to be significantly greater, over the agerange, 20-60 years, for iliac arteries (-100 by/yr, P=0.01) than foriliac veins (47 bp/yr, P=0.14). See U.S. Pat. No. 5,489,508, issued Feb.2, 1996. This decrease in mean TRF for plaque regions versus plaque-freeregions of medial tissue from the same blood vessel is consistent withaugmented cell turnover of tissue associated with atheroscleroticplaques. These results indicate that telomere length is a biomarker forcell turnover and proliferative capacity in tissues associated withcardiovascular disease, including cells of intimal and medial tissues.

Telomere length can be used not only as a biomarker for a diseasecondition but also as a prognostic indicator of disease stage. Forexample, in one study, telomere length measurements of CD28-CD8+ cellsof HIV-infected subjects had significantly shorter TRF lengths thanthose of uninfected controls. In addition, the telomere lengthmeasurements of the lymphocyte subset of CD28-CD8+ cells compared toCD28+CD8+ cells in HIV-infected individuals for all subjects studiedwere consistently shorter. In fact, the mean TRF lengths of CD28-CD8+cells of the HIV-infected subjects (5-7 kb) were similar to thoseobserved for centenarians and for senescent T-cell cultures. Becauseloss of telomeric DNA is a marker of cell division, telomere shorteningin CD8+ cells can be attributed to extensive cell division or turnover.Therefore, telomere shortening in the CD8+ cells can be ascribed toimmune exhaustion that results from chronic-immune system activation andas such can be an indicator of HIV disease progression. Thus, telomerelength in all cells such as CD8+ cells, CD4+ cells, and other cells ofthe immune system can be used for prognosis of the course of HIVinfection or AIDS.

After a disease is diagnosed, telomere length measurement can be used todetermine whether the disease is at an early or late stage of diseaseprogression. For leukemia, telomere length is indicative of the timesince disease onset and the relative rate of abnormal cellproliferation. Leukemic cells that have been dividing at increased ratesfor long periods of time have shorter telomeres than normal bone marrowcells. There is a progressive decrease in mean TRF length in blood andbone marrow leukocytes during the course of chronic lymphoid leukemia(CLL): the average TRF lengths in one study of CLL patients were: normalindividuals (controls) 10.0-16.0 kb; early-stage CLL, 7.9 kb; and latestage CLL, 4.4 kb (Counter et al., 85 Blood 2315, 1995).

In chronic myeloid leukemia (CML) patients, there is a wide variation inTRF of bone marrow cells, i.e., the TRF is 2.8-12.8 kb. In one study of44 CML patients, nine had a mean TRF length within the age-matchednormal range. The remainder of the patients had short telomeres (average5.6 kb) as compared to those of age-matched normal peripheral bloodmononuclear cells. Those patients with shorter TRF lengths at the timeof diagnosis experienced a shorter interval until blast crisis andresponded less well to treatment than did patients with normal TRF.Telomere length depends upon the number of cell divisions and so, asillustrated in chronic CML patients, represents a new marker of diseasestate. CML patients having leukocytes with normal TRFs may be at anearly stage of the disease and thus respond better to therapy.

In a similar fashion, the mean TRF was much shorter in the leukocytes ofacute myeloid leukemia (AML) patients than in control leukocytes of bonemarrow and peripheral blood from normal individuals. In addition, blastcells from seven AML patients had shorter telomeres than bloodmononuclear cells isolated during remission (Yamada et al., 95 J. Clin.Invest. 1117, 1995). Thus, the presence of leukocytes (or other cells)with short TRF length is indicative of late stage or acute phase diseasein these leukemias.

Telomere length measurement can also be used to diagnose fertilityproblems. In one study, telomere length was measured in sperm cells fromboth fertile and infertile males. Sperm cells from certain infertilemales had significantly shorter telomeres than did sperm cells from thefertile males.

The methods of the invention have application in determining theproliferative capacity of a tissue as well as individual cells or celltypes within a tissue. Many tissues regenerate from only a small numberof stem cells. With in situ hybridization, one can identify andquantitate telomere length in such stem cells on an individual as wellas collective basis. These methods allow one to determine telomerelength on a chromosome-by-chromosome basis and to evaluateinterchromosomal variance of telomere length. These measurements aremade by quantitating the fluorescent intensity of bound probe for eachindividual cell nucleus or chromosome using confocal microscopy or flowcytometry analysis. Flow cytometry provides the added benefit ofallowing the sorting of cells or chromosomes based on telomere lengthand cell type or chromosome identity (i.e., X chromosome separated fromthe Y and all other chromosomes). These sorted cells or chromosomes canthen used as desired, i.e., for manipulation and/or subsequenttherapeutic reintroduction into cell or, individual. These and otherapplications of the invention are further elaborated in the Examplesbelow.

EXAMPLES

The following examples describe specific aspects of the invention toillustrate the invention and to provide a description of the methods forthose of skill in the art. The examples should not be construed aslimiting the invention, as the examples merely provide specificmethodology useful in understanding and practicing the invention.

EXAMPLE 1 Dot-Blot Method for Measuring Telomere Length

This example describes a dot-blot method for measuring telomere length.Telomere length is determined by correlating the signal intensity ofprobe bound to the telomere region to telomere length. To facilitateunderstanding of the method, a number of different DNA preparation stepsthat can be used in the process are described. In addition, althoughsolution compositions are provided, those of skill in the art willrecognize that variations of these solutions can readily be made byappropriately modifying the concentrations of the various components in,as well as the composition of, the solution.

Two six-well plates of 293 cells seeded at 100,000 cells/well and onesix-well plate of 293 seeded cells at 75,000 cells/well were washedtwice with cold phosphate buffered saline solution (1×PBS is composed of10 mM K₃ PO₄ and 150 mM NaCl) to remove residual growth media. Cellmembranes were lysed by adding 1.5 ml of extraction buffer (extractionbuffer is 10 mM Tris, pH 8.0, 0.1M ethylenediaminotetraacetic acid(EDTA), pH 8.0, 0.1M NaCl, 0.5% sodium dodecyl sulfate (SDS), and 100μg/ml proteinase K) to each well and incubating the samples at 50° C.for 3-16 hours; an additional aliquot (15 μl) of 10 mg/ml proteinase Kwas typically added after 1 hour.

After this incubation, the cellular RNA in the sample was degraded byadding 15 μl of 500 μg/ml DNase-free RNase to each well and incubatingthe samples for an additional hour at 37° C. The lysed cell extracts(referred to below as "DNA stock solutions") were then removed from thewells and transferred to tubes. The tubes were then heated at 65° C. for10-20 minutes (at this point, the solutions can be snap frozen for lateruse). Alternate or additional DNA preparation steps can be used, e.g.,enzymatic digestion (i.e., with proteolytic, RNase, or restrictionenzymes), phenol extraction, and/or ethanol (EtOH) precipitation.

To demonstrate linearity of signal intensity to DNA concentration,aliquots of the DNA stock solutions, 150 μl, 75 μl, 37.5 μl, and 18.75μl, were transferred from the tubes and spotted into individual wells of96-well plates (Costar). The DNA was denatured in a solution of 0.4MNaOH and 10 mM EDTA, and the contents were transferred to 96-well filterplates. The filters were rinsed with 160 μl of 0.4M NaOH, vacuumfiltered, rinsed with a solution of 0.3M NaCl and 0.03M sodium citrate(2×SSC) to remove the denaturing solution, and dried.

³² P-labeled riboprobe was prepared by combining in a sterile Eppendorf™tube 50 μl of 5×transcription buffer (purchased from Stratagene), 10 μlof HindIII-digested pBLRep4 (1 μg/μl, purchased from Stratagene), 10 μlof 10 mM rATP, 10 μl of 10 mM rCTP, 10 μl of 10 mM rGTP, 10 μl of 0.75Mdithiothreitol (DTT, purchased from Stratagene), 50 μl of ³² P-UTP (0.25mCi), 2 μl of T3 RNA Polymerase (purchased from Stratagene), and 98 μlof DEPC-treated water and incubating in a 37° C. water bath for 30minutes. Plasmid pBLRep4 comprises 100 telomere repeat sequences(5'-TTAGGG-3') inserted into the EcoRl site of the plasmidpBluescriptIISK+ (Stratagene). After incubation, the tube was pulse spunin a Pico Fuge™ (Stratagene) and then 10 μl of RNase free DNase I(Boehringer Mannheim) was added, followed by incubation at 37° C. for 15minutes. To the resultant mixture was added 260 μl ofphenol/chloroform/isoamyl alcohol (PCIA, 26:25:1 ratio); the solutionwas vortexed and then certrifuged in a Pico Fuge™ (Stratagene) forapproximately 4 minutes. After centrifugation, the top aqueous layer istransferred into another sterile, Eppendorf tube into which is alsoadded approximately 26 μl of 3M sodium acetate. The reaction is mixedand then approximately 650 μl of 200 proof ethanol is added with mixing.The probe is precipitated out by maintaining the temperature of the tubeat -20° C. for at least 30 minutes. The tube is then centrifuged for 10minutes in the Pico Fuge™ (Stratagene). The supernatant is discarded andthe pellet is resuspended in 100 μl of 1×TE buffer (1×TE buffer iscomposed of 10 mM Tris and 1 mM EDTA). The total volume in the tube isthen brought up to 1 ml with 1×TE buffer. The resuspended probe can bestored at -20° C. until used.

The DNA on the filters was then hybridized with 50 μl (50 μCi/μg DNA) ofthe ³² P-UTP labeled riboprobe (comprising repeats of the sequence5'-CCCTAA-3', as discussed above) in hybridization buffer (hybridizationbuffer is composed of 6×SSC, 1×Denhardt's solution, 20 mM sodiumphosphate, pH 7.2, and 0.4% SDS) overnight at 65° C. The filters werewashed four to five times in a wash solution composed of 1×SSC and 0.1%SDS and then exposed for at least 1 hour in a PhosphoImager cassette(Molecular Dynamics) and scanned. FIG. 2 shows the results presentedgraphically. The signal intensity increased with the amount of DNA, asdemonstrated by this graph.

Known concentrations of DNA samples prepared from various cells (OVCAR4, OVCAR 3, OVCAR 5, OVCAR 8, and SKOV-3) were analyzed with this methodto measure differences in telomere length. As a comparison, the TRFlengths of these cells were also determined using a conventional method.Table 1 shows that signal intensity generally increased with increasedTRF using the dot-blot method.

                                      TABLE 1                                     __________________________________________________________________________    Dot-Blot Method: Signal Intensity and TRF Length                              Sample                                                                             TRF(kb)                                                                            2 μg DNA                                                                        1 μg DNA                                                                        0.5 μg DNA                                                                       0.25 μg DNA                                                                       0.125 μg DNA                              __________________________________________________________________________    OVCAR 5                                                                            2.39 NT    851742                                                                            439777                                                                              211908 104927                                       OVCAR 3                                                                            3.55 2701170                                                                            1326068                                                                            608263                                                                              247371 NT                                           OVCAR 4                                                                            4.89 3235960                                                                            1600895                                                                            688627                                                                              262075 NT                                           OVCAR 8                                                                            7.51 2901921                                                                            1406973                                                                            633572                                                                              267539 NT                                           SK-OV-3                                                                            10.69                                                                              5612527                                                                            2880505                                                                            1359104                                                                             512160 NT                                           __________________________________________________________________________     NT  not tested.                                                          

For example, SK-OV-3 cells, which have the greatest TRF of the cells inthese samples (10.69 kb), also exhibited the strongest signal using thedot-blot method at each concentration tested.

These data show, as predicted, that the TRF length also includes atleast a portion of the length of the subtelomeric region. Using thismethod, one can calculate the length of the subtelomeric region includedin the TRF length. This ability to distinguish subtelomeric lengthversus 5'-TTAGGG-3' telomeric repeat length has important implicationsin disease therapy and prognosis. Because the signal strength generatedby this method is a more accurate indicator of replicative capacity thanTRF length, this method can be used to determine which patients have abetter prognosis and to determine the required duration of treatments,such as telomerase inhibition treatment. For example, as demonstrated inTable 1, OVCAR 8 comprises a shorter telomeric repeat region than OVCAR4; however, the TRF of OVCAR 8 is greater, indicating that the TRFlength for OVCAR 8 includes a greater portion of the subtelomeric regionthan that of OVCAR 4. Because the telomeric repeat length is indicativeof the remaining proliferative capacity of the cell, a cancer patientwith a tumor composed of cells like the OVCAR 8 cells would require ashorter period of treatment with a telomerase inhibitor than a patientwith a tumor composed of cells such as OVCAR 4 cells, even though TRFlength analysis would suggest otherwise. While the telomere lengthmeasurements undertaken in this example were for immortal cell lines,this method can also be used for mortal cell lines as a measure ofreplicative capacity.

EXAMPLE 2 Slot-Blot Method for Measuring Telomere Length

This example illustrates use of the slot-blot method for measuringtelomere length. In this process, the DNA is cross-linked to a solidphase prior to hybridization with a probe.

About 1.5 μg of total genomic DNA from BJ cells (human foreskinfibroblast) and S2C cells (human skin fibroblast) at different PDL wascompletely digested overnight with a mixture of restriction enzymesEcoRI and HindIII. In addition, 1.25 μg of plasmid pBLRep4 was digestedwith restriction enzyme XbaI and used as a standard control. This amountof the plasmid is equivalent to 52.6 pmol of the DNA consisting of5'-TTAGGG-3' repeats. The negative control, 1.5 μg of Lambda phage DNA,was digested with restriction enzymes Sau3A and HindIII and used tocalculate the background signal.

The digested DNA was extracted with phenol/chloroform, precipitated withethanol, and then resuspended in 100 μl of 1×TE buffer (1×TE buffer iscomposed of 10 mM Tris and 1 mM EDTA). To this solution was added andmixed thoroughly 200 μl of 5×SSC.

A Schleicher & Schuell Minifold II™ apparatus was used to prepare thefilters employed in the slot blot method. The blotting paper was wettedwith 5×SSC, and each slot of the apparatus was rinsed with about 300 μlof 5×SSC. The digested DNA samples were then loaded into designatedslots. To generate a standard curve on the blot, the digested plasmidpBLRep4 was diluted serially, and 0.009375 μg, 0.00625 μg, 0.003125 μg,and 0.0015625 μg were then loaded onto separate slots. All liquid wasvacuum filtered through the blot; the DNA was retained on the blot. Theblot was then removed and placed on a piece of 3 MM Whatman, paper (VWR)and air dried for 30 minutes.

The DNA was denatured by placing the blot on a stack of 3 MM papersoaked with a solution of 0.5N NaOH and 1.5M NaCl for 30 minutes. TheDNA was then neutralized by placing the blot on 3 MM paper soaked with asolution of 0.5M Tris (pH 7.4) and 1.5M NaCl for 30 minutes. Followingneutralization, the DNA was cross-linked to the blot with a UVSTRATALINKER 1800™ device (Stratagene). Then, the DNA was incubated in15 ml of prehybridization buffer (prehybridization buffer is composed of5×SSC, 5×Denhardt's solution (1 g of Ficoll (Type 400, Pharmacia), 1 gof polyvinylpyrrolidone, 1 g of bovine serum albumin (Fraction V, Sigma,and sufficient water to make 100 ml), 0.02M phosphate (pH 6.5), 0.1mg/ml salmon sperm DNA, 0.5% SDS, and 50% formamide) for 2 hours. Thistreatment was followed by hybridization of the DNA in 15 ml ofprehybridization buffer containing ³² P-labeled5'-TTAGGGTTAGGGTTAGGG-3'(SEQ. ID NO.:13) probe (1 million cpm/ml), andincubation overnight at 37° C.

After probe hybridization, the blot was washed once with 500 ml of asolution composed of 1×SSC and 0.1% SDS for 10 minutes at roomtemperature, then washed twice with 500 ml of the same wash solution at37° C. for 20 minutes, and then placed in a PhosphoImager™ detectorovernight. The signal intensity of the hybridized probe was thenanalyzed.

The signal intensity of probe hybridization in cells at different PDLwas converted to a relative number that reflected the average length oftelomeres according to the standard curve generated using the pBLRep4control. The results, shown in Table 2, demonstrated generally adecrease in telomere length with increased PDL. The results correlatewell with the corresponding TRF lengths and signal strength determinedby conventional methods (see FIG. 3).

                  TABLE 2                                                         ______________________________________                                        Slot-Blot Method: PDL, Signal Intensity ("SI"), and Relative                  Signal Intensity ("RSI")                                                                                  Control                                           BJ Cells     S2C Cells      pBLRep4                                           PDL  SI      RSI     PDL  SI    RSI   (pmol) SI                               ______________________________________                                        27.5 357338  100     30   286761                                                                              100    0.06576                                                                              76976                           28.6 346486  97      39   258953                                                                              90    0.132  155074                           41.2 280452  71      44   262724                                                                              91    0.263  285200                           46.6 293800  81      47   209496                                                                              71    0.394  402212                           57.2 270484  74      53   159030                                                                              52                                            69.2 219510  59      62   157456                                                                              52                                            72.2 230505  62      69.2 166010                                                                              54                                            73.5 194970  53      71.2 150172                                                                              49                                            80.0 219628  59      73.2 174636                                                                              58                                            88.2 119028  29                                                               91.4 113223  28                                                               ______________________________________                                    

The results in Table 2 show generally that bound probe signal intensitydecreases with increased PDL. In addition, similar results were obtainedfor BJ cells and S2C cells, and signal intensity increased withincreased amounts of the control, pBLRep4. The pBLRep4 plasmid resultscan be used to produce a standard curve generated by graphing knownamounts of telomere repeat sequence versus the corresponding signalintensity determined using the slot-blot method. A standard curvegenerated using the data for pBLRep4 in Table 2 shows a linear increasein signal with increased amounts of pBLRep4.

EXAMPLE 3 Flow Cytometry for Telomere Length Quantitation

This example illustrates a method for measuring telomere length of thechromosomes of an individual cell. The method can be performedsimultaneously with the analysis of cellular DNA content. Thismethodology also allows one to sort cells or chromosomes based ontelomere length.

Growing cells are harvested by trypsinization and washed in PBS(phosphate-buffered saline), as per standard procedures. The washedcells are then fixed by adding freshly made, cold (4° C.) 3:1 100%anhydrous methanol:glacial acetic acid to the resuspended cell pelletwith gentle mixing. Cells can be stored at 4° C. prior to analysis.

Alternatively, if one desires to measure the telomere length of aspecific chromosome, the cells can be lysed to release intact nuclei,which are then fixed by incubation overnight in 2% paraformaldehyde (pH7.0). Lysing the cells is accomplished by suspending the cells in 20 mMNaCl, 10 mM MgCl₂, 20 mM Tris (pH 7.2); incubating the cell suspensionat 37° C. for 5 minutes; adding an equal volume of triton X-100 andmixing. For convenience, only analysis of intact cells, as opposed tonuclei, is described below. Those of skill in the art will recognizethat this method is equally applicable to the analysis of telomeres ofspecific chromosomes.

Prior to hybridization, the cells are centrifuged and washed three timeswith PBS. Cells are then treated with RNase A for 20 minutes at 37° C.(100 μg/ml in PBS) followed by pepsin treatment (1 mg/ml, pH 2.0) for 5minutes at 37° C.

The cells are centrifuged and then resuspended in hybridization buffercomposed of 70% deionized formamide containing FITC-labeled PNA probe,sonicated salmon sperm DNA (or other commercially available reagent toprevent non-specific probe hybridization), and 10 mM Tris (pH 7.2), atroom temperature for 2-8 hours. Because all cells fluoresce, a controlexperiment is conducted under the same conditions, except that the PNAprobe is unlabeled to determine background fluorescence.

After the hybridization step, the cells are washed to remove unboundprobe. The cells are washed three times in a solution composed of 70%formamide, 10 mM Tris (pH 7.2), and 0.05% triton X-100. The cells arethen resuspended in PBS and analyzed on a flow cytometer.

Alternatively, a labeled DNA probe can be used in place of the PNAprobe. If a DNA probe is used, the following procedure is implementedafter fixing the cells. The DNA is denatured by adding 0.5 ml of asolution consisting of 70% deionized formamide and 2×SSC to the cellpellet (˜1 million cells) and heating to 70° C. in a water bath for 2-5minutes. The cells are then cooled on ice and centrifuged. The cellpellet is kept on ice until adding the probe to prevent renaturation ofthe DNA. The labeled DNA probe (i.e., biotin or digoxigenin) issuspended in 250 μL of hybridization solution (70% formamide, 2×SSC,2×Denhardt's solution, 10% dextran sulfate, 50 mM Tris, pH 7.5), heatedat 75°-80° C. for 10 minutes to denature the DNA, and cooled on ice. Thecooled probe is added to the cell pellet and hybridized at 37° C.overnight. The cells are then washed in 50% formamide and 2×SSC at roomtemperature, collected by centrifugation, then resuspended in a solutioncontaining streptavidin-FITC or a FITC-labeled anti-digoxigeninantibody. The resultant mixture is incubated at room temperature for onehour and the cells are collected by centrifugation and washedthoroughly. A control can be performed with steptavidin lacking the FITClabel or employing a non-labeled antibody in the procedure.

The cells are analyzed using a flow cytometer. A standard optics andfilter arrangement for a FITC-generated signal is used (488 nmexcitation, 525 nm bandpass filter for emission). The signals to becollected include log and linear FITC fluorescence (525 nm) and lightscatter (0° angle and 90° angle) as correlated parameters.

During flow cytometry, the cell passes a laser at a wavelength whichgenerates scattered light and fluorescence signals from the cell. Thephotomultiplier tube detects the generated photons, and the signal ispassed through a digital-to-analog converter. The signal can beamplified. The resultant signals can be displayed either linearly orlogarithmically. Logarithmic displays provide better separation of thepeaks, whereas linear displays generally provide more sensitivity.

The cells can also be counterstained with a DNA specific dye such aspropidium iodide (PI) to measure cellular DNA content simultaneously. Ifcounterstaining is used, the same filter set-up as described above isused (the PI signal is measured using a 610 nm bandpass filter). Thisset-up will allow determination of cell cycle position and cellular DNAcontent, as well as quantitation of the hybridized probe signal. Theintensity of signal from bound probe per chromosome or cell isproportional to the number of telomeric repeats and to the telomerelength. As the signal intensity is measured, the instrument can beprogrammed to deflect the cells into specific tubes based upon thesignal and the corresponding telomere length.

EXAMPLE 4 PCR-Based Telomere Measurement

This example describes a PCR-based method for measuring telomere length.The telomeric DNA is first treated with an exonuclease to generate bluntends, and then, a double-stranded linker is attached to the 3' end ofthe telomere. A forward primer complementary to the linker and asubtelomeric return primer complementary to the subtelomeric region ofchromosome X and Y are extended by PCR in the presence of nucleotidetriphosphates. The long PCR primer extension products are then separatedby size on a gel, and size standards on the gel are used to determinetelomere length.

Genomic DNA is digested with Bal31 nuclease (4 U/μg DNA, BoehringerMannheim) for 5 minutes at 30° C. to remove modified nucleotides at theends of the telomeres and to blunt-end the DNA. Following the digestion,the Bal31 nuclease is inactivated by the addition of 0.2Methylenebis(oxyethylenenitrilo)-tetraacetic acid (EGTA) to a finalconcentration of 15 mM. The DNA is then extracted usingphenol/chloroform, precipitated with ethanol, and resuspended in 1×T4DNA polymerase buffer (13×T4 DNA polymerase buffer is composed of 50 mMNaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM dithiothreitol, pH 7.9) to afinal DNA concentration of 0.1-0.5 μg/μl. To improve blunt-endingefficiency, the DNA is treated with T4 DNA polymerase (New EnglandBiolabs) in a mixture comprising 0.25 mM dNTPs (0.0625 mM each of DATP,dGTP, dCTP and dTTP) for 30 minutes at 37° C. The T4 DNA polymerase isinactivated by heating for 15 minutes at 65° C. Alternative nucleasesand DNA polymerases can be substituted in this reaction, i.e., Mung Beannuclease or Pfu DNA polymerase. In addition, the Bal31 treatment of theDNA can be eliminated; and the DNA can be treated directly with T4 DNApolymerase in the presence of dNTPs to generate blunt ends.

The double-stranded linker SLIC-II/aSLIC is prepared by adding equimolaramounts of the phosphorylated SLIC-II oligonucleotide,5'-GGAATTCTGGTCGACGGATCCTGA-3' (SEQ. ID NO.:8), and thenon-phosphorylated complementary oligonucleotide aSLIC,3'-CCTTAAGACCAGCTGCCTAGGACT-5' (SEQ. ID NO.:9), at room temperature,heating to 94° C. for 3-4 minutes, placing the reaction vesselcontaining the oligonucleotides in a water bath pre-heated to 65°-70°C., and cooling the vessel to room temperature. The annealing efficiencyis checked by digesting 0.5 pmol of the prepared linker with EcoRIfollowed by electrophoretic analysis (15% polyacrylamide gel) of thedigestion products and an untreated linker control. The ratio of thesingle-stranded, double-stranded, and digested double-strandedoligonucleotide bands on the gel provides a measure of the efficiency ofthe annealing process and a check that annealing is in the correctregister.

The double-stranded linker is ligated onto the blunt ends of the DNA bycombining, at 16° C., the blunt-ended DNA (0.1-0.5 μg), thephosphorylated linker (SLIC-II/aSLIC, 1 μg), deionized water (17 μl),10×blunt-end ligation buffer containing 10 mM ATP (2 μl, New EnglandBiolabs), and T4 DNA ligase (1 μl, 400 U, New England Biolabs). Thereaction is allowed to proceed for 6-8 hours and then quenched by heatinactivation of the T4 DNA ligase for 15 minutes at 65° C. As a positivecontrol to determine ligation efficiency, a radioactively labeledSLIC-II/aSLIC linker is prepared as above using ³² P-labeled SLIC-II andligated to T4 DNA polymerase-treated, MboI digested DNA from BJ cellsand/or HinfIlRsaI digested DNA from BJ cells.

PCR amplification of the telomeric region is accomplished by combining0.1 μg of DNA ligated to SLIC II/aSLIC; 5 μl of 2.5 mM dNTPs (0.0625 mMeach of dATP, dGTP, dCTP and dTTP) solution; 5 μl of 10×long PCR buffer(20 mM Tris·HCl (pH 9.0), 150 μg/ml BSA, 3.5 mM MgCl₂, and 16 mM (NH₄)₂SO₄); 40 pmol of revXpJCTN primer (5'-CTGATWGGTCCACTTTCAGAGGG-3', (SEQ.ID NO.:7)), 1 μl of ExTaqTM DNA polymerase (Oncor); and deionized waterto a total volume of 50 μl. The reaction vessel is then transferred to athermal cycler for 30 cycles, each cycle comprising incubationtemperatures and periods of 94° C. for 1 min. and 65° C. for 1 min. anda final incubation at 72° C. for 10 min.

The amplified primer extension products are resolved on a 0.5% agarosegel. The telomere length is determined by comparison of the primerextension products with size standards.

The reagents employed in the examples are commercially available or canbe prepared using commercially available instrumentation, methods, orreagents known in the art. The foregoing examples illustrate variousaspects of the invention and practice of the methods of the invention.The examples are not intended to provide an exhaustive description ofthe many different embodiments of the invention. Thus, although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, thoseof ordinary skill in the art will realize readily that many changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 13                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      TGCAATTATTTTACTATCTGTTATCGG27                                                 (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      TGACCTGTTTTAAAGAGTATGCTCAG26                                                  (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CCCTCTGAAAGTGGACCWATCAG23                                                     (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      CTTTTATTCTCTAATCTGCTCCC23                                                     (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      TAGGGGTTGTCTCAGGGTCCTA22                                                      (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GGGAGCAGATTAGAGAATAAAAG23                                                     (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      CTGATWGGTCCACTTTCAGAGGG23                                                     (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      GGAATTCTGGTCGACGGATCCTGA24                                                    (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      TCAGGATCCGTCGACCAGAATTCC24                                                    (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     CCGTCGACCAGAATTCC17                                                           (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     CAGGATCCGTCGACCAG17                                                           (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     CCCTAACCCTAACCCTAACCCTAA24                                                    (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     TTAGGGTTAGGGTTAGGG18                                                          __________________________________________________________________________

We claim:
 1. A method for measuring telomere length, said methodcomprising the steps of:(a) contacting denatured chromosomal DNA thathas not been fractionated by gel electrophoresis with a labeled probehaving a sequence complementary to a telomere repeat sequence underconditions such that said probe hybridizes specifically to telomericDNA; (b) measuring amount of bound probe; and (c) correlating saidamount of bound probe measured relative to a control of known telomerelength with telomere length.
 2. The method of claim 1, wherein said DNAis immobilized on a solid support.
 3. The method of claim 1, whereinprior to step (b) said DNA is digested with a nuclease.
 4. The method ofclaim 1, wherein said telomere is a human telomere.
 5. The method ofclaim 1, wherein said chromosomal DNA is human chromosomal DNA.
 6. Themethod of claim 5, wherein said chromosomal DNA is from a blood sample.7. The method of claim 5, wherein said chromosomal DNA is from a tissuesample.
 8. The method of claim 5, wherein said chromosomal DNA is from asperm sample.
 9. The method of claim 5, wherein said chromosomal DNA isfrom a urine sample.